ISO 11737-3:2023

NORME ISO
INTERNATIONALE 11737-3
Première édition
2023-06
Stérilisation des produits de santé —
Méthodes microbiologiques —
Partie 3:
Essai des endotoxines bactériennes
Sterilization of health care products — Microbiological methods —
Part 3: Bacterial endotoxin testing
Numéro de référence
ISO 11737-3:2023(F)
© ISO 2023

---------------------- Page: 1 ----------------------
ISO 11737-3:2023(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2023
Tous droits réservés. Sauf prescription différente ou nécessité dans le contexte de sa mise en œuvre, aucune partie de cette
publication ne peut être reproduite ni utilisée sous quelque forme que ce soit et par aucun procédé, électronique ou mécanique,
y compris la photocopie, ou la diffusion sur l’internet ou sur un intranet, sans autorisation écrite préalable. Une autorisation peut
être demandée à l’ISO à l’adresse ci-après ou au comité membre de l’ISO dans le pays du demandeur.
ISO copyright office
Case postale 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Genève
Tél.: +41 22 749 01 11
E-mail: copyright@iso.org
Web: www.iso.org
Publié en Suisse
ii
  © ISO 2023 – Tous droits réservés

---------------------- Page: 2 ----------------------
ISO 11737-3:2023(F)
Sommaire Page
Avant-propos .v
Introduction . vi
1 Domaine d'application .1
1.1 Inclusions. 1
1.2 Exclusions . 1
2 Références normatives .1
3 Termes et définitions . 1
4 Exigences générales . .7
5 Sélection des produits . 8
5.1 Généralités . 8
5.2 Sélection des unités de produits . 9
6 Méthodes pour l'EEB .10
6.1 Généralités . 10
6.2 Prise en compte d'une limite d'endotoxines applicable . 10
6.2.1 Limite d'endotoxines. 10
6.2.2 Calcul de la limite d'endotoxines pour la solution d'extrait . 11
6.2.3 Dilution maximale significative (DMS ou MVD). 11
6.3 Paramètres d'essai critiques . 12
6.3.1 Température .12
6.3.2 Temps.12
6.3.3 pH .12
6.4 Équipements et matériaux .12
6.5 Réactifs .12
7 Validation de la méthode pour l'EEB (validation de l'EEB) .13
7.1 Généralités . 13
7.2 Validation du produit et de la méthode d'essai . 14
7.2.1 Technique par gélification. 14
7.2.2 Méthodes cinétiques et en point final (techniques colorimétriques et
turbidimétriques) . 14
7.3 Préparation des échantillons . 15
7.3.1 Généralités .15
7.3.2 Produits de santé solides . 15
7.3.3 Produits de santé à base de solution aqueuse . 16
7.3.4 Interférence de l'échantillon . 16
7.4 Qualification des réactifs et des analystes . 16
7.4.1 Qualification des réactifs de la technique par gélification . 16
7.4.2 Qualification des réactifs des méthodes cinétiques et des méthodes en
point final . 17
7.4.3 Qualification de l'analyste . 17
8 Essais de routine, surveillance et interprétation des données .17
8.1 Essais de routine . 17
8.1.1 Essai limite par gélification . 17
8.1.2 Dosage par gélification . 18
8.1.3 Méthodes cinétiques et en point final (colorimétriques et turbidimétriques) . 18
8.2 Surveillance (fréquence d'essai) . 18
8.3 Interprétation des résultats . 19
8.3.1 Généralités . 19
8.3.2 Méthodes par gélification . 19
8.3.3 Méthode cinétique et méthode en point final . 20
8.4 Analyse des données .20
8.5 Méthodes statistiques . 20
iii
© ISO 2023 – Tous droits réservés

---------------------- Page: 3 ----------------------
ISO 11737-3:2023(F)
9 Maintenance de la méthode d'EEB .20
9.1 Généralités . 20
9.2 Modifications apportées au produit ou au procédé de fabrication, ou aux deux.20
9.3 Modifications de la méthode d'EEB . 20
10 Alternatives aux essais par lots .21
10.1 Généralités . 21
10.2 Critères pour établir des alternatives aux essais par lots . 21
10.3 Évaluation du procédé de fabrication . 22
10.3.1 Planification de la qualité des procédés de fabrication .22
10.3.2 Conception du procédé .22
10.3.3 Contrôle du procédé . 22
10.4 Maîtrise des modifications .22
10.5 Mise à jour de l'évaluation des risques . 23
Annexe A (informative) Recommandations relatives aux essais des endotoxines
bactériennes (suivant les paragraphes du présent document) .24
Annexe B (informative) Historique et contexte de l'essai des endotoxines bactériennes
(EEB) .45
Annexe C (informative) Recommandations relatives aux résultats de limites hors
spécification (OSL) et aux investigations associées .50
Annexe D (informative) Recommandations relatives à la surveillance des procédés de
fabrication ou aux essais des composants .55
Annexe E (informative) Recommandations relatives à la réalisation d'une évaluation des
risques pour étayer les alternatives aux essais par lots .58
Annexe F (informative) Répartition type des responsabilités .63
Bibliographie .65
iv
  © ISO 2023 – Tous droits réservés

---------------------- Page: 4 ----------------------
ISO 11737-3:2023(F)
Avant-propos
L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes
nationaux de normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est
en général confiée aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude
a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,
gouvernementales et non gouvernementales, en liaison avec l'ISO participent également aux travaux.
L'ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui
concerne la normalisation électrotechnique.
Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont
décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier, de prendre note des différents
critères d'approbation requis pour les différents types de documents ISO. Le présent document
a été rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2
(voir www.iso.org/directives).
L’ISO attire l’attention sur le fait que la mise en application du présent document peut entraîner
l’utilisation d’un ou de plusieurs brevets. L’ISO ne prend pas position quant à la preuve, à la validité
et à l’applicabilité de tout droit de brevet revendiqué à cet égard. À la date de publication du présent
document, l’ISO n'avait pas reçu notification qu’un ou plusieurs brevets pouvaient être nécessaires à sa
mise en application. Toutefois, il y a lieu d’avertir les responsables de la mise en application du présent
document que des informations plus récentes sont susceptibles de figurer dans la base de données de
brevets, disponible à l'adresse www.iso.org/brevets. L’ISO ne saurait être tenue pour responsable de ne
pas avoir identifié tout ou partie de tels droits de propriété.
Les appellations commerciales éventuellement mentionnées dans le présent document sont données
pour information, par souci de commodité, à l’intention des utilisateurs et ne sauraient constituer un
engagement.
Pour une explication de la nature volontaire des normes, la signification des termes et expressions
spécifiques de l'ISO liés à l'évaluation de la conformité, ou pour toute information au sujet de l'adhésion
de l'ISO aux principes de l'Organisation mondiale du commerce (OMC) concernant les obstacles
techniques au commerce (OTC), voir www.iso.org/iso/fr/avant-propos.
Le présent document a été élaboré par le comité technique ISO/TC 198, Stérilisation des produits de
santé.
Une liste de toutes les parties de la série ISO 11737 se trouve sur le site web de l'ISO.
Il convient que l’utilisateur adresse tout retour d’information ou toute question concernant le présent
document à l’organisme national de normalisation de son pays. Une liste exhaustive desdits organismes
se trouve à l’adresse www.iso.org/fr/members.html.
v
© ISO 2023 – Tous droits réservés

---------------------- Page: 5 ----------------------
ISO 11737-3:2023(F)
Introduction
Un pyrogène est une substance susceptible de provoquer de la fièvre. La recherche de pyrogène est
nécessaire pour la libération de nombreux produits de santé. Les pyrogènes peuvent être classés
en deux groupes: microbiens (par exemple, bactéries, champignons, virus) et non microbiens (par
exemple, médicaments, matériaux des dispositifs, stéroïdes, fractions de plasma, voir la série de normes
ISO 10993). Les contaminants pyrogènes prédominants observés lors de la fabrication de produits
de santé sont les endotoxines bactériennes, qui sont des composants de la membrane cellulaire des
bactéries à Gram-négatif. Bien que les bactéries à Gram-positif, les champignons et les virus puissent
être pyrogènes, ils agissent par des mécanismes différents (effets systémiques) et à un degré moindre
que les bactéries à Gram-négatif. Seul l'essai des endotoxines bactériennes à Gram-négatif (EEB)
utilisant des réactifs de lysat d'amébocytes de Limulus polyphemus ou de Tachypleus tridentatus est
couvert dans le présent document. Les autres méthodologies de détection des endotoxines, telles que
l'activation des monocytes et le facteur C recombinant (rFc), ne sont pas incluses (voir B.12) dans le
présent document.
Les endotoxines sont les composants lipopolysaccharides (LPS) de poids moléculaire situés dans la
membrane cellulaire externe des bactéries à Gram-négatif, qui peuvent provoquer de la fièvre, une
méningite et une chute rapide de la pression artérielle s'ils sont introduits dans la circulation sanguine
ou dans certains autres tissus de l'organisme. Les composants de la membrane cellulaire externe, qui
sont principalement composés de protéines, de phospholipides et de LPS, sont constamment libérés par
la cellule dans le milieu environnant. Les endotoxines sont omniprésentes dans la nature, stables et
suffisamment petites pour passer à travers les filtres de stérilisation conventionnels. Les procédés de
stérilisation inactivent les micro-organismes sur ou dans les produits, mais n'inactivent généralement
pas les endotoxines sur les produits. Or, des procédés contrôlés permettent d'éviter la contamination
par des endotoxines.
L'absence de pyrogène sur un produit de santé peut être obtenue par les moyens suivants:
a) des techniques de fabrication qui empêchent ou contrôlent la contamination par des endotoxines
(par exemple la contamination par des bactéries à Gram-négatif);
b) la dépyrogénation par inactivation des endotoxines (par exemple, chaleur sèche) ou élimination
physique (par exemple, rinçage, distillation, ultrafiltration).
L'objectif du présent document consiste à décrire les exigences et les recommandations relatives aux
essais des endotoxines bactériennes. Ces essais portent notamment sur les produits devant être non
pyrogènes en raison de leur usage prévu ou de la mention «non pyrogène» sur l'étiquette, ou des deux.
Des recommandations sont également formulées au sujet de la sélection des unités de produit, de la
validation des méthodes, de l'utilisation des techniques pour les essais de routine, de l'interprétation
des résultats d'essai, et des alternatives aux essais par lots et à l'évaluation des risques. Les annexes
présentent des informations concernant:
— les recommandations relatives aux essais des endotoxines bactériennes (Annexe A);
— l'historique et le contexte relatifs aux essais EEB (Annexe B);
— les recommandations relatives aux résultats de limites hors spécification (OSL) et aux investigations
associées (Annexe C);
— les recommandations relatives à la surveillance des procédés de fabrication ou aux essais des
composants (Annexe D);
— les recommandations relatives à la réalisation d'une évaluation des risques pour étayer les
alternatives aux essais par lots (Annexe E);
— la répartition type des responsabilités (Annexe F).
Le présent document est fondé sur l'ANSI/AAMI ST72. Plusieurs sections du présent document ont été
réorganisées et étendues ou modifiées par rapport à l'ANSI/AAMI ST72.
vi
  © ISO 2023 – Tous droits réservés

---------------------- Page: 6 ----------------------
NORME INTERNATIONALE ISO 11737-3:2023(F)
Stérilisation des produits de santé — Méthodes
microbiologiques —
Partie 3:
Essai des endotoxines bactériennes
1 Domaine d'application
1.1 Inclusions
Le présent document spécifie les critères généraux à appliquer pour la détermination des endotoxines
bactériennes présentes sur ou dans les produits de santé, les composants ou les matières premières
en utilisant les méthodes d'essai des endotoxines bactériennes (EEB), à l'aide des réactifs de lysat
d'amébocytes.
1.2 Exclusions
1.2.1 Le présent document ne s'applique pas à l'évaluation des pyrogènes autres que les endotoxines
bactériennes. Les autres méthodologies de détection des endotoxines ne sont pas incluses (voir B.12).
1.2.2 Le présent document ne traite pas de l'établissement de spécifications particulières de limite
d'endotoxines.
2 Références normatives
Le présent document ne contient aucune référence normative.
3 Termes et définitions
Pour les besoins du présent document, les termes et définitions suivants s'appliquent.
L'ISO et l'IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en
normalisation, consultables aux adresses suivantes:
— ISO Online browsing platform: disponible à l'adresse https:// www .iso .org/ obp
— IEC Electropedia: disponible à l'adresse https:// www .electropedia .org/
3.1
essai des endotoxines bactériennes
EEB (BET, bacterial endotoxins test)
essai destiné à mesurer les endotoxines bactériennes en combinant un échantillon pour essai aqueux
ou un extrait d'échantillon pour essai avec un lysat d'amébocytes de Tachypleus (TAL) (3.41) ou un lysat
d'amébocytes de limule (LAL) (3.28) comme réactif et en mesurant la réaction proportionnelle qui en
résulte par des techniques visuelles, turbidimétriques (3.42) ou colorimétriques (3.3)
1
© ISO 2023 – Tous droits réservés

---------------------- Page: 7 ----------------------
ISO 11737-3:2023(F)
3.2
lot
quantité donnée de produit, destinée ou censée être de nature et de qualité uniformes, et qui a été
fabriquée pendant un cycle de fabrication spécifié
[SOURCE: ISO 11139:2018, 3.21]
3.3
technique colorimétrique
méthodologie de l'essai des endotoxines bactériennes (EEB) (3.1) qui quantifie les endotoxines sur la base
d'une réaction colorée mesurée, proportionnelle à l'interaction entre le lysat d'amébocytes de limule
(LAL) (3.28) et l'endotoxine
3.4
endotoxine standard de contrôle
CSE
préparation d'endotoxine standard dont le titre a été normalisé par rapport à l'endotoxine standard de
référence (RSE, Reference Standard Endotoxin) (3.37) pour un lot spécifique de lysat d'amébocytes de
limule (LAL) (3.28)
3.5
dépyrogénation
procédé utilisé pour éliminer ou désactiver des substances pyrogènes jusqu'à un niveau spécifié
Note 1 à l'article: Les substances pyrogènes incluent les endotoxines bactériennes.
[SOURCE: ISO 11139:2018, 3.77]
3.6
contact direct
dispositif médical ou composant de dispositif médical qui entre en contact physique avec un tissu de
l'organisme
[SOURCE: ISO 10993-1:2018, 3.6]
3.7
produit fini
échantillons ou produits arrivés au terme de l'ensemble du procédé de fabrication
Note 1 à l'article: Aux fins du présent document, l'analyse du produit fini peut être effectuée avant la stérilisation
(échantillons de pré-stérilisation) ou après la stérilisation (échantillons de post-stérilisation). Pour les limites,
voir 5.2.6.
3.8
endotoxine
endotoxine bactérienne
lipopolysaccharide (LPS) (3.29) de la paroi cellulaire d'une bactérie à Gram-négatif qui se caractérise
par sa stabilité à la chaleur et qui provoque diverses réactions inflammatoires chez l'Homme et chez les
animaux
[SOURCE: ISO 11139:2018, 3.101]
3.9
limite d'endotoxines
quantité maximale admissible d'endotoxines présente sur le produit ou dans une solution d'extraction
du produit
2
  © ISO 2023 – Tous droits réservés

---------------------- Page: 8 ----------------------
ISO 11737-3:2023(F)
3.10
unité d'endotoxines
UE
unité internationale
UI
unité de mesure normalisée de l'activité de l'endotoxine initialement établie par rapport à l'activité
contenue dans 0,2 ng du lot EC-2 de l'endotoxine standard de référence (RSE) (3.37) (matériau standard
de référence de la pharmacopée américaine (USP))
Note 1 à l'article: Actuellement, l'endotoxine RSE EC-6, le lot G de l'USP, et l'endotoxine standard internationale
primaire (IS) de l'Organisation mondiale de la santé sont des sous-lots de la même préparation d'endotoxine, ce
[45]
qui rend l'UE et l'UI égales .
3.11
point final
concentration la plus faible d'une solution d'essai ou de contrôle pour laquelle une réaction positive à
l'endotoxine bactérienne est observée
Note 1 à l'article: Cette définition est utilisée pour les essais des endotoxines bactériennes dépendant de la
concentration, par contraste avec les méthodes en point final dépendant de la dilution décrites en A.6.1.1.
3.12
activation
anomalie de l'essai des endotoxines bactériennes (EEB) (3.1) caractérisée par le fait qu'un facteur non lié
à l'endotoxine, généralement dû à une caractéristique de l'échantillon pour essai, provoque une réaction
supérieure à la quantité d'endotoxines présente
3.13
technique par gélification
méthodologie de l'essai des endotoxines bactériennes (EEB) (3.1) qui quantifie ou détecte l'endotoxine
sur la base d'une réaction de production de caillots proportionnelle à l'interaction entre le lysat
d'amébocytes de limule (LAL) (3.28) et l'endotoxine
3.14
moyenne géométrique au point final
antilogarithme de la moyenne des valeurs logarithmiques par rapport aux points finaux (3.11) des séries
de réplicats de dilution converties en un nombre en base 10 utilisé pour établir la tendance centrale ou
la valeur typique d'une solution d'essai
3.15
produit de santé
dispositif médical, pouvant être un dispositif médical de diagnostic in vitro, ou produit médicinal,
notamment un produit biopharmaceutique
[SOURCE: ISO 11139:2018, 3.132]
3.16
contact indirect
dispositif médical ou composant de dispositif médical par lequel passe un fluide ou un gaz, avant que
le fluide ou le gaz entre en contact physique avec un tissu de l'organisme (dans ce cas, le dispositif
médical ou le composant de dispositif médical lui-même n'entre pas en contact physique avec le tissu de
l'organisme)
[SOURCE: ISO 10993-1:2018, 3.11]
3.17
inhibition
anomalie de l'essai des endotoxines bactériennes (EEB) (3.1) caractérisée par le fait qu'un facteur non lié
à l'endotoxine, généralement dû à une caractéristique de l'échantillon pour essai, provoque une réaction
inférieure à la quantité d'endotoxines présente
3
© ISO 2023 – Tous droits réservés

---------------------- Page: 9 ----------------------
ISO 11737-3:2023(F)
3.18
validation de la méthode
essai d'inhibition/d'activation
essai servant à déterminer si un échantillon particulier contient des facteurs d'interférence qui
diminuent sa précision par l'introduction d'une activation (3.12) ou d'une inhibition (3.17) dans le
système d'essai
3.19
interférence
facteur d'interférence observé au cours de l'essai qui dépasse le seuil acceptable pour une technique
d'essai des endotoxines bactériennes (EEB) (3.1) donnée (par exemple, un contrôle positif du produit
indiquant un niveau d'endotoxines détecté inférieur à 50 % ou supérieur à 200 % ou ±2 lambda)
3.20
intraoculaire
situé ou survenant à l'intérieur de l'œil ou administré par ce dernier
3.21
facteurs d'interférence
facteur indépendant de l'endotoxine, généralement dû à une caractéristique de l'échantillon pour essai,
qui provoque une inhibition (3.17) ou une activation (3.12)
3.22
intravasculaire
situé ou survenant à l'intérieur du cœur ou administré par le cœur ou les vaisseaux sanguins
3.23
intralymphatique
situé ou survenant à l'intérieur du vaisseau lymphatique ou administré par ce dernier
3.24
intrathécal
situé ou survenant à l'intérieur de l'espace sous la membrane arachnoïde du cerveau ou de la moelle
épinière, ou administré par ce dernier
3.25
méthode cinétique
techniques photométriques quantitatives (turbidimétriques ou colorimétriques) pour l'essai des
endotoxines bactériennes (EEB) (3.1)
3.26
matériau réactif au LAL
LAL-MR
matériau réactif au lysat d'amébocytes de limule
tout composé autre qu'une endotoxine qui activera la cascade de coagulation du lysat d'amébocytes de
limule (LAL) (3.28) et provoquera une activation (3.12)
3.27
lambda
λ
sensibilité reve
...

Deleted: © ISO 2022 – All rights
reserved…
ISO/FDIS 11737-3
Deleted: 2022-12-31¶
ISO/TC 198
Deleted: -
Deleted: :2022(X)
Secretariat: ANSI
Deleted: /WG 8
Date: 2023-01-31
Deleted: AAMI (for
Deleted: )
Sterilization of health care products — Microbiological
methods —
Part 3:
Bacterial endotoxin testing
Stérilisation des dispositifs médicaux — Méthodes microbiologiques —
Partie 3: Essai des endotoxines bactériennes
FDIS stage
© ISO 2023 – All rights reserved

---------------------- Page: 1 ----------------------
ISO/FDIS 11737-3:2023(E)
Deleted: 2022
© ISO 2023
Deleted: 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this
publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical,
including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can
be requested from either ISO at the address below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
E-mail: copyright@iso.org
Deleted: Fax: +41 22 749 09 47¶
Website: www.iso.org
Email
Published in Switzerland
Deleted: 2022
ii © ISO 2023 – All rights reserved

---------------------- Page: 2 ----------------------
ISO/FDIS 11737-3:2023(E)
Deleted:
Contents Deleted: 2022
Foreword . Error! Bookmark not defined.
Introduction. Error! Bookmark not defined.
1 Scope . Error! Bookmark not defined.
1.1 Inclusions . Error! Bookmark not defined.
1.2 Exclusions . Error! Bookmark not defined.
2 Normative references . Error! Bookmark not defined.
3 Terms and definitions . Error! Bookmark not defined.
4 General requirements . Error! Bookmark not defined.
5 Selection of products . Error! Bookmark not defined.
5.1 General . Error! Bookmark not defined.
5.2 Selection of product units . Error! Bookmark not defined.
6 Methods for BET . Error! Bookmark not defined.
6.1 General . Error! Bookmark not defined.
6.2 Consideration of an applicable endotoxin limit. Error! Bookmark not defined.
6.3 Critical test parameters . Error! Bookmark not defined.
6.4 Equipment and materials . Error! Bookmark not defined.
6.5 Reagents . Error! Bookmark not defined.
7 Method suitability for BET (BET validation) . Error! Bookmark not defined.
7.1 General . Error! Bookmark not defined.
7.2 Product and test method suitability . Error! Bookmark not defined.
7.3 Sample preparation . Error! Bookmark not defined.
7.4 Reagent and analyst qualification. Error! Bookmark not defined.
8 Routine testing, monitoring and interpretation of data . Error! Bookmark not defined.
8.1 Routine testing . Error! Bookmark not defined.
8.2 Monitoring (test frequency) . Error! Bookmark not defined.
8.3 Interpretation of results . Error! Bookmark not defined.
8.4 Data analysis . Error! Bookmark not defined.
8.5 Statistical methods . Error! Bookmark not defined.
9 Maintenance of the BET method . Error! Bookmark not defined.
9.1 General . Error! Bookmark not defined.
9.2 Changes to either the product or manufacturing process, or bothError! Bookmark not defined.
9.3 Changes to the BET method . Error! Bookmark not defined.
10 Alternatives to batch testing . Error! Bookmark not defined.
(informative) Guidance on bacterial endotoxin testing (following the subclauses in this
document) . Error! Bookmark not defined.
(informative) History and background on the bacterial endotoxins test (BET)Error! Bookmark not
defined.
(informative) Guidance on out of specified limits (OSL) and failure investigationError! Bookmark
not defined.
(informative) Guidance on in-process monitoring of manufacturing processes or component
testing . Error! Bookmark not defined.
Deleted: 2022
© ISO 2023 – All rights reserved iii

---------------------- Page: 3 ----------------------
ISO/FDIS 11737-3:2023(E)
Deleted: 2022
(informative) Guidance on conducting a risk assessment to support alternatives to batch testing
Error! Bookmark not defined.
(informative) Typical assignment of responsibilities . Error! Bookmark not defined.
Bibliography . Error! Bookmark not defined.

Deleted: 2022
iv © ISO 2023 – All rights reserved

---------------------- Page: 4 ----------------------
ISO/FDIS 11737-3:2023(E)
Deleted:
Foreword
Deleted: 2022
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO
collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any
patent rights identified during the development of the document will be in the Introduction and/or on
the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the World
Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 198, Sterilization of health care products.
A list of all parts in the ISO 11737 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
Field Code Changed
Deleted: 2022
© ISO 2023 – All rights reserved v

---------------------- Page: 5 ----------------------
ISO/FDIS 11737-3:2023(E)
Deleted: 2022
Introduction
A pyrogen is any substance that can induce fever. Testing for pyrogens is required for release of many
health care products. Pyrogens can be classified into two groups: microbial (e.g. bacteria, fungi, viruses)
and non-microbial (e.g. drugs, device materials, steroids, plasma fractions; see the ISO 10993 series). The
predominant pyrogenic contaminants encountered in the manufacturing of health care products are
bacterial endotoxins, which are components of the cell walls of Gram-negative bacteria. Although Gram-
positive bacteria, fungi, and viruses can be pyrogenic, they do so through different mechanisms (systemic
effects) and to a lesser degree than Gram-negative bacteria. Only the Gram-negative bacterial endotoxins
test (BET) using amebocyte lysate reagents from Limulus polyphemus or Tachypleus tridentatus is covered Deleted: are
in this document. Other endotoxin detection methodologies, such as monocyte activation and
recombinant Factor C (rFc), are not included (see B.12) in this document. Deleted: B.12).
Endotoxins are the molecular weight lipopolysaccharide (LPS) components of the outer cell wall of Gram-
negative bacteria, that can cause fever, meningitis, and a rapid fall in blood pressure if introduced into
the blood stream or certain other tissues of the body. The outer cell wall components, which are
composed primarily of proteins, phospholipids and LPS, are constantly released by the cell into the
Deleted: ,
surrounding environment. Endotoxins are ubiquitous in nature, stable, and small enough to pass through
conventional sterilizing filters. Sterilization processes will inactivate microorganisms on or in products,
but usually do not inactivate endotoxin on products. With controlled processes, endotoxin contamination
can be prevented.
The non-pyrogenicity of a health care product can be achieved through the following:
a) manufacturing techniques that prevent or control endotoxin contamination (e.g. contamination with
Deleted: a)
Gram-negative bacteria);
Deleted: ),
b) depyrogenation by endotoxin inactivation (e.g. dry heat) or physical removal (e.g. rinsing,
Deleted: b)
distillation, ultrafiltration).
The purpose of this document is to describe the requirements and guidance for testing for bacterial
endotoxins. This includes product required to be non-pyrogenic based on either intended use or non-
pyrogenic label claim, or both. Guidance is also provided on selection of product units, method suitability,
use of techniques for routine testing, interpretation of test results, and alternatives to batch testing and
risk assessment. Information on the following is provided in the annexes:
— guidance on bacterial endotoxin testing (Annex A);
Deleted: —
Deleted: (Annex A);
— the history and background on the BET (Annex B);
Deleted: —
— guidance on out of specified limits (OSL) and failure investigation (Annex C);
Deleted: (Annex B);
Deleted: —
— guidance on in-process monitoring of manufacturing or component testing (Annex D);
Deleted: (Annex C);
— guidance on conducting a risk assessment to support alternatives to batch testing (Annex E);
Deleted: —
Deleted: (Annex D);
— typical assignment of responsibilities (Annex F).
Deleted: —
This document is based on ANSI/AAMI ST72. Several sections in this document have been restructured
Deleted: (Annex E);
and extended or changed from ANSI/AAMI ST72.
Deleted: —
Deleted: (Annex F).
Deleted: 2022
vi © ISO 2023 – All rights reserved

---------------------- Page: 6 ----------------------
ISO/FDIS 11737-3:2023(E)
Sterilization of health care products — Microbiological methods —
Part 3:
Bacterial endotoxin testing
1 Scope
1.1 Inclusions
This document specifies general criteria to be applied in the determination of bacterial endotoxins on or
in health care products, components or raw materials using bacterial endotoxins test (BET) methods,
Deleted: ,
using amebocyte lysate reagents.
1.2 Exclusions
1.2.1 This document is not applicable to the evaluation of pyrogens other than bacterial endotoxins.
Other endotoxin detection methodologies are not included (see B.12).
Deleted: B.12).
1.2.2 This document does not address setting specific endotoxin limit specifications.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
Deleted: —
— IEC Electropedia: available at https://www.electropedia.org/
Deleted: —
3.1
bacterial endotoxins test
BET
assay for measuring bacterial endotoxins by combining an aqueous test sample or test sample extract
with Tachypleus amebocyte lysate (TAL) (3.41) or Limulus amebocyte lysate (LAL) (3.28) reagent and Deleted: (3.41)
measuring the resulting proportional reaction via visual, turbidimetric or chromogenic techniques (3.3)
Deleted: (3.27)
Deleted: ,
3.2
Deleted: (3.3)
batch
defined quantity of a product intended or purported to be uniform in character and quality produced
during a specified cycle of manufacture
[SOURCE: ISO 11139:2018, 3.21]
3.3
chromogenic technique
bacterial endotoxins test(BET) (3.1) methodology that quantifies endotoxins on the basis of a measured Deleted: (3.1)
colour-producing reaction proportional to the interaction of Limulus amebocyte lysate (LAL) (3.28) and
Deleted: (3.28)
endotoxin
3.4
control standard endotoxin
CSE
Deleted: (3.37)
endotoxin standard preparation whose potency has been standardized against the Reference Standard
Deleted: (3.28)
Endotoxin (RSE) (3.37) for a specific batch of Limulus amebocyte lysate (LAL) (3.28)
Deleted: 2022
© ISO 2023 – All rights reserved 1

---------------------- Page: 7 ----------------------
ISO/FDIS 11737-3:2023(E)
3.5
depyrogenation
process used to remove or deactivate pyrogenic substances to a specified level
Note 1 to entry: Pyrogenic substances include bacterial endotoxins.
[SOURCE: ISO 11139:2018, 3.77]
3.6
direct contact
medical device or medical device component that comes into physical contact with body tissue
[SOURCE: ISO 10993-1:2018, 3.6]
Deleted: -
3.7
end product
product samples that have completed the entire manufacturing process
Note 1 to entry: For the purposes of this document, end-product testing can be performed prior to sterilization (pre-
sterilization samples) or after sterilization (post-sterilization samples). For limitations see 5.2.6.
Deleted: 5.2.6.
3.8
endotoxin
bacterial endotoxin
lipopolysaccharide (LPS)(3.29) component of the cell wall of Gram-negative bacteria that is heat stable
and elicits a variety of inflammatory responses in animals and humans
[SOURCE: ISO 11139:2018, 3.101]
3.9
endotoxin limit
maximum allowable amount of endotoxin present on the product or in a product extraction solution
3.10
endotoxin unit
EU
international unit
IU
standard unit of measure for endotoxin activity initially established relative to the activity contained in
0,2 ng of the Reference Standard Endotoxin (RSE) (3.37) Lot EC-2 [US Pharmacopeia (USP) standard Deleted: .
reference material]
Deleted: US
Deleted: (3.37)
Note 1 to entry: Currently, the US RSE EC-6, USP Lot G, and the World Health Organization’s primary international
[45]
endotoxin standard (IS) are sub-lots of the same endotoxin preparation, making the EU and IU equal . Deleted: (
Deleted: )
3.11
[45]
Deleted: .
end point
most dilute concentration of a test or control solution for which a positive reaction for bacterial endotoxin
is observed
Note 1 to entry: This definition is used for concentration dependent bacterial endotoxin testing, in contrast to
dilution dependent end point methods described in A.6.1.1.
Deleted: A.6.1.1.
3.12
enhancement
bacterial endotoxins test (BET) (3.1) anomaly in which a non-endotoxin related factor, usually attributable Deleted: (3.1)
to a characteristic of the test sample, elicits a test reaction greater than the amount of endotoxin present
Deleted: 2022
2 © ISO 2023 – All rights reserved

---------------------- Page: 8 ----------------------
ISO/FDIS 11737-3:2023(E)
3.13
gel-clot technique
bacterial endotoxins test (BET) (3.1) methodology that quantifies or detects endotoxin on the basis of a Deleted: (3.1)
clot-producing reaction proportional to the interaction of Limulus amebocyte lysate (LAL) (3.28) and
Deleted: (3.28)
endotoxin
3.14
geometric mean end point
antilog of the average of the logarithmic values with respect to the end points (3.11) from replicate Deleted: (3.11)
dilution series converted back to a base 10 number used to establish the central tendency or typical value
from a test solution
3.15
health care product
medical device, including in vitro diagnostic medical device, or medicinal product, including
biopharmaceutical
[SOURCE: ISO 11139:2018, 3.132]
3.16
indirect contact
medical device or medical device component through which a fluid or gas passes, prior to the fluid or gas
coming into physical contact with body tissue (in this case the medical device or medical device
component itself does not physically contact body tissue)
[SOURCE: ISO 10993-1:2018, 3.11] Deleted: -
3.17
inhibition
bacterial endotoxins test (BET) (3.1) anomaly in which a non-endotoxin related factor, usually attributable
Deleted: (3.1)
to a characteristic of the test sample, elicits a test reaction less than the amount of endotoxin present
3.18
method suitability
inhibition/enhancement test
Deleted: (method suitability)
test used to determine whether a particular sample contains interfering factors that diminish its accuracy
by introducing enhancement (3.12) or inhibition (3.17) into the test system
Deleted: (3.12)
Deleted: (3.17)
3.19
interference
interfering factor observed in the performance of the test that exceeds the acceptable threshold for a
given bacterial endotoxins test (BET) (3.1) technique (e.g. positive product control that indicates a Deleted: (3.1)
detected endotoxin level less than 50 % or greater than 200 % or ±2 lambda)
3.20
intraocular
Deleted: , adj.
located or occurring within or administered through the eye
3.21
interfering factors
non-endotoxin related factor, usually attributable to a characteristic of the test sample, that causes
inhibition (3.17) or enhancement (3.12) Deleted: (3.17)
Deleted: (3.12)
3.22
intravascular Deleted: , adj.
located or occurring within or administered through the heart or blood vessels
3.23
Deleted: , adj.
intralymphatic
located or occurring within or administered through a lymph vessel
Deleted: 2022
© ISO 2023 – All rights reserved 3

---------------------- Page: 9 ----------------------
ISO/FDIS 11737-3:2023(E)
3.24
intrathecal, adj,
located, or occurring within or administered through the space under the arachnoid membrane of the
brain or spinal cord
3.25
kinetic method
photometric quantitative techniques (turbidimetric or chromogenic) for bacterial endotoxins test (BET)
(3.1) Deleted: (3.1)
3.26
LAL reactive material
LAL-RM
Deleted: (
Limulus amebocyte lysate reactive material
Deleted: )
any non-endotoxin compound that will activate the Limulus amebocyte lysate (LAL) (3.28) clotting
Deleted: (3.28)
cascade and cause enhancement (3.12)
Deleted: (3.12)
3.27
lambda
λ
labelled sensitivity of a Limulus amebocyte lysate (LAL) (3.28) gel-clot reagent, expressed in EU/mL or,
Deleted: labeled
for chromogenic or turbidimetric tests, the lowest point (endotoxin concentration) on the referenced
Deleted: (3.28)
standard curve
3.28
Limulus amebocyte lysate
LAL
reagent extracted from amebocytes taken from hemolymph of the horseshoe crab, Limulus polyphemus,
that reacts with endotoxin, to form a gelatinous clot and is used to estimate endotoxin levels in bacterial
endotoxins test (BET) (3.1) methods Deleted: (3.1)
Note 1 to entry: The term LAL is sometimes used to describe Tachypleus amebocyte lysate (TAL) (3.41), as both are
Deleted: (3.41),
similar lysates that are used in the BET. They also are often generically referred to as “lysate”.
3.29
lipopolysaccharide
LPS
Gram-negative bacterial cell wall component composed of lipid A, a core polysaccharide, and an O-side
chain
3.30
maximum valid dilution
MVD
maximum amount a sample can be diluted, or the total extraction volume used relative to the sensitivity
of a bacterial endotoxins test (BET) (3.1) in which the specified endotoxin limit (3.9) can be detected Deleted: (3.1)
Deleted: (3.9)
3.31
medical device
instrument, apparatus, implement, machine, appliance, implant, reagent for in vitro use, or software Deleted: ,
material or other similar or related article, intended by the manufacturer to be used, alone or in
combination, for human beings, for one or more of the specific medical purpose(s) of:
— diagnosis, prevention, monitoring, treatment or alleviation of disease;
Deleted: —
— diagnosis, monitoring, treatment, alleviation of or compensation for an injury;
Deleted: —
— investigation, replacement, modification or support of the anatomy or of a physiological process;
Deleted: —
— supporting or sustaining life;
Deleted: —
Deleted: 2022
4 © ISO 2023 – All rights reserved

---------------------- Page: 10 ----------------------
ISO/FDIS 11737-3:2023(E)
— control of conception;
Deleted: —
— disinfection of medical devices;
Deleted: —
— providing information by means of in vitro examination of specimens derived from the human body;
Deleted: —
and does not achieve its primary intended action by pharmacological, immunological or metabolic means,
but which may be assisted in its intended function by such means
Note 1 to entry: Products which can be considered to be medical devices in some jurisdictions, but not in others
Deleted: may
include:
— items specifically intended for cleaning or sterilization of medical devices;
Deleted: —
— pouches, reel goods, sterilization wrap, and reusable containers for packaging of medical devices for
Deleted: —
sterilization;
— disinfection substances;
Deleted: —
— aids for persons with disabilities;
Deleted: —
— devices incorporating either animal or human tissues, or both;
Deleted: —
Deleted: and/
— devices for in vitro fertilization or assisted reproduction technologies.
Deleted: —
[SOURCE: ISO 13485:2016, 3.11, modified — The first two list items in the Note 1 to entry have been
added. In Note 1 to entry, "may be considered" has been changed to "can be considered" to indicate
possibility rather than permission.]
3.32
non-pyrogenic
Deleted: , adj.
not inducing a fever
Note 1 to entry: Describes an item or product that contains endotoxin levels that conform to specified limits.
Deleted: comply
3.33
out of specified limits
OSL
sample with a valid bacterial endotoxins test (BET) (3.1) result that exceeds a product endotoxin limit (3.9) Deleted: (3.1)
specification
Deleted: (3.9)
Note 1 to entry: The term OSL applies only within the context of this document and does not imply compliance with
any other regulatory guidance dealing with out of specification (OOS) results.
3.34
product positive control
PPC
sample spiked with a known amount of endotoxin used for confirmation that the product being tested is
not subject to interfering factors (3.21) Deleted: (3.21)
3.35
pyrogen
substance that induces a fever
3.36
pyrogenic
Deleted: , adj.
inducing a fever
Note 1 to entry: Describes an item or product that contains endotoxin levels above specified limits.
Deleted: 2022
© ISO 2023 – All rights reserved 5

---------------------- Page: 11 ----------------------
ISO/FDIS 11737-3:2023(E)
3.37
Reference Standard Endotoxin
RSE
US Pharmacopeia (USP) endotoxin reference standard that has a defined potency of 10,000 USP EUs per
vial
3.38
repeat test
analysis of additional product samples from a previously tested batch or another batch
3.39
retest
reanalysis of previously tested product samples or product sample preparation
3.40
standard control series
serial dilution series of Reference Standard Endotoxin (RSE) (3.37) or control standard endotoxin (CSE) Deleted: (3.37)
(3.4) used to verify Limulus amebocyte lysate (LAL) (3.28) sensitivity
Deleted: (3.4)
Deleted: (3.28)
3.41
Tachypleus amebocyte lysate
TAL
reagent extracted from amebocytes taken from hemolymph of the horseshoe crab, Tachypleus tridentatus,
which reacts with endotoxin, to form a gelatinous clot and is used to estimate endotoxin levels in bacterial
endotoxins test (BET) (3.1) methods Deleted: (3.1)
Note 1 to entry: The term TAL is sometimes used to describe Limulus amebocyte lysate (LAL) (3.28), as both are
Deleted: (3.28),
similar lysates that are used in the BET. They also are often generically referred to as “lysate”.
3.42
turbidimetric technique
bacterial endotoxins test (BET) (3.1) methodology that quantifies or detects endotoxin on the basis of a Deleted: (3.1)
measured turbidity reaction proportional to the interaction of Limulus amebocyte lysate (LAL) (3.28) and
Deleted: (3.28)
endotoxin
3.43
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended
use or application have been fulfilled
Note 1 to entry: The objective evidence needed for a validation is the result of a test or other form of determination
such as performing alternative calculations or reviewing documents.
Note 2 to entry: The word “validated” is used to designate the corresponding status.
Note 3 to entry: The use conditions for validation can be real or simulated.
[SOURCE: ISO 9000:2015, 3.813]
3.44
verification
confirmation, through the provision of objective evidence, that specified requirements have been fulfilled
Note 1 to entry: The objective evidence needed for a verification can be the result of an inspection or of other forms
of determination such as performing alternative calculations or reviewing documents.
Note 2 to entry: The word “verified” is used to designate the corresponding status.
[SOURCE: ISO 9000:2015, 3.8.12, modified — The original Note 2 to entry has been deleted and Note 3
Deleted: accordingly
has been renumbered as Note 2 to entry.]
Deleted: 2022
6 © ISO 2023 – All rights reserved

---------------------- Page: 12 ----------------------
ISO/FDIS 11737-3:2023(E)
3.45
water for bacterial endotoxins test
WBET
purified water employable as a solvent, diluent, and/or extractant that is non-reactive with the lysate
employed at the detection limit of the reagent, and does not elicit interference (3.19) with methodology Deleted: (3.19)
in use [typically Limulus amebocyte lysate (LAL) (3.28) reagent water, water for injection, or other
Deleted: (
appropriate solution meeting these requirements]
Deleted: )
4 General requirements
4.1 The development, validation and routine control of products with acceptable endotoxin levels are
critical elements in the realization of some types of health care products. To ensure the consistent
implementation of the requirements specified in this document, the necessary processes shall be Deleted: need to
established, implemented and maintained. Processes of particular importance in relation to the
development, validation and routine endotoxin control of a process include but are not limited to:
— control of documentation, including records,
Deleted: —
— assignment of management responsibility,
Deleted: —
— provision of adequate resources, including competent human resources and infrastructure,
Deleted: —
— control of product provided by external parties,
Deleted: —
— identification and traceability of product throughout the process, and
Deleted: —
— control of non-conforming product.
Deleted: —
NOTE ISO 13485 covers all stages of the life cycle of medical devices in the context of quality management
systems for regulatory purposes. National and/or regional regulatory requirements for the provision of health care
product can require the implementation of a full quality management system and the assessment of that system by
a recognized conformity assessment body.
4.2 A process shall be specified for the calibration of all equipment, including instrumentation for test
purposes, used in meeting the
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 11737-3
ISO/TC 198
Sterilization of health care products —
Secretariat: ANSI
Microbiological methods —
Voting begins on:
2023-02-15
Part 3:
Voting terminates on:
Bacterial endotoxin testing
2023-04-12
Stérilisation des dispositifs médicaux — Méthodes
microbiologiques —
Partie 3: Essai des endotoxines bactériennes
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/FDIS 11737-3:2023(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. © ISO 2023

---------------------- Page: 1 ----------------------
ISO/FDIS 11737-3:2023(E)
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 11737-3
ISO/TC 198
Sterilization of health care products —
Secretariat: ANSI
Microbiological methods —
Voting begins on:
Part 3:
Voting terminates on:
Bacterial endotoxin testing
Stérilisation des dispositifs médicaux — Méthodes
microbiologiques —
Partie 3: Essai des endotoxines bactériennes
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
RECIPIENTS OF THIS DRAFT ARE INVITED TO
ISO copyright office
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
CP 401 • Ch. de Blandonnet 8
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
CH-1214 Vernier, Geneva
DOCUMENTATION.
Phone: +41 22 749 01 11
IN ADDITION TO THEIR EVALUATION AS
Reference number
Email: copyright@iso.org
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/FDIS 11737-3:2023(E)
Website: www.iso.org
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
Published in Switzerland
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
ii
  © ISO 2023 – All rights reserved
NATIONAL REGULATIONS. © ISO 2023

---------------------- Page: 2 ----------------------
ISO/FDIS 11737-3:2023(E)
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
1.1 Inclusions. 1
1.2 Exclusions . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General requirements . 7
5 Selection of products .8
5.1 General . 8
5.2 Selection of product units . 8
6 Methods for BET . 9
6.1 General . 9
6.2 Consideration of an applicable endotoxin limit . 10
6.2.1 Endotoxin limit . 10
6.2.2 Calculation of endotoxin limit for the extract solution . 10
6.2.3 Maximum valid dilution (MVD) . 10
6.3 Critical test parameters . 11
6.3.1 Temperature . 11
6.3.2 Time . 11
6.3.3 pH . 11
6.4 Equipment and materials . 11
6.5 Reagents .12
7 Method suitability for BET (BET validation) .12
7.1 General .12
7.2 Product and test method suitability .12
7.2.1 Gel-clot technique .12
7.2.2 Kinetic and end point methods (chromogenic and turbidimetric techniques) .13
7.3 Sample preparation . 14
7.3.1 General . 14
7.3.2 Solid health care products . 14
7.3.3 Aqueous health care products . 15
7.3.4 Sample interference .15
7.4 Reagent and analyst qualification . 15
7.4.1 Gel-clot technique reagent qualification . 15
7.4.2 Kinetic and end point method reagent qualification .15
7.4.3 Analyst qualification . 16
8 Routine testing, monitoring and interpretation of data .16
8.1 Routine testing . 16
8.1.1 Gel-clot limit test . 16
8.1.2 Gel-clot assay . 16
8.1.3 Kinetic and end point methods (chromogenic and turbidimetric) . 17
8.2 Monitoring (test frequency) . 17
8.3 Interpretation of results . 17
8.3.1 General . 17
8.3.2 Gel clot methods. 18
8.3.3 Kinetic and end point methods . 18
8.4 Data analysis . 18
8.5 Statistical methods . 18
9 Maintenance of the BET method .18
9.1 General . 18
iii
© ISO 2023 – All rights reserved

---------------------- Page: 3 ----------------------
ISO/FDIS 11737-3:2023(E)
9.2 Changes to either the product or manufacturing process, or both . 18
9.3 Changes to the BET method . 19
10 Alternatives to batch testing .19
10.1 General . 19
10.2 Criteria for establishing alternatives to batch testing . 19
10.3 Manufacturing process assessment . 20
10.3.1 Quality planning of manufacturing processes . 20
10.3.2 Process design .20
10.3.3 Process control . 20
10.4 Change control . 21
10.5 Maintenance of risk assessment . 21
Annex A (informative) Guidance on bacterial endotoxin testing (following the subclauses
in this document) .22
Annex B (informative) History and background on the bacterial endotoxins test (BET) .42
Annex C (informative) Guidance on out of specified limits (OSL) and failure investigation .46
Annex D (informative) Guidance on in-process monitoring of manufacturing processes or
component testing .50
Annex E (informative) Guidance on conducting a risk assessment to support alternatives
to batch testing .53
Annex F (informative) Typical assignment of responsibilities .58
Bibliography .60
iv
  © ISO 2023 – All rights reserved

---------------------- Page: 4 ----------------------
ISO/FDIS 11737-3:2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 198, Sterilization of health care products.
A list of all parts in the ISO 11737 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
© ISO 2023 – All rights reserved

---------------------- Page: 5 ----------------------
ISO/FDIS 11737-3:2023(E)
Introduction
A pyrogen is any substance that can induce fever. Testing for pyrogens is required for release of many
health care products. Pyrogens can be classified into two groups: microbial (e.g. bacteria, fungi, viruses)
and non-microbial (e.g. drugs, device materials, steroids, plasma fractions; see the ISO 10993 series).
The predominant pyrogenic contaminants encountered in the manufacturing of health care products
are bacterial endotoxins, which are components of the cell walls of Gram-negative bacteria. Although
Gram-positive bacteria, fungi, and viruses can be pyrogenic, they do so through different mechanisms
(systemic effects) and to a lesser degree than Gram-negative bacteria. Only the Gram-negative bacterial
endotoxins test (BET) using amebocyte lysate reagents from Limulus polyphemus or Tachypleus
tridentatus is covered in this document. Other endotoxin detection methodologies, such as monocyte
activation and recombinant Factor C (rFc), are not included (see B.12) in this document.
Endotoxins are the molecular weight lipopolysaccharide (LPS) components of the outer cell wall of
Gram-negative bacteria, that can cause fever, meningitis, and a rapid fall in blood pressure if introduced
into the blood stream or certain other tissues of the body. The outer cell wall components, which are
composed primarily of proteins, phospholipids and LPS, are constantly released by the cell into the
surrounding environment. Endotoxins are ubiquitous in nature, stable, and small enough to pass
through conventional sterilizing filters. Sterilization processes will inactivate microorganisms on or
in products, but usually do not inactivate endotoxin on products. With controlled processes, endotoxin
contamination can be prevented.
The non-pyrogenicity of a health care product can be achieved through the following:
a) manufacturing techniques that prevent or control endotoxin contamination (e.g. contamination
with Gram-negative bacteria);
b) depyrogenation by endotoxin inactivation (e.g. dry heat) or physical removal (e.g. rinsing,
distillation, ultrafiltration).
The purpose of this document is to describe the requirements and guidance for testing for bacterial
endotoxins. This includes product required to be non-pyrogenic based on either intended use or
non-pyrogenic label claim, or both. Guidance is also provided on selection of product units, method
suitability, use of techniques for routine testing, interpretation of test results, and alternatives to batch
testing and risk assessment. Information on the following is provided in the annexes:
— guidance on bacterial endotoxin testing (Annex A);
— the history and background on the BET (Annex B);
— guidance on out of specified limits (OSL) and failure investigation (Annex C);
— guidance on in-process monitoring of manufacturing or component testing (Annex D);
— guidance on conducting a risk assessment to support alternatives to batch testing (Annex E);
— typical assignment of responsibilities (Annex F).
This document is based on ANSI/AAMI ST72. Several sections in this document have been restructured
and extended or changed from ANSI/AAMI ST72.
vi
  © ISO 2023 – All rights reserved

---------------------- Page: 6 ----------------------
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 11737-3:2023(E)
Sterilization of health care products — Microbiological
methods —
Part 3:
Bacterial endotoxin testing
1 Scope
1.1 Inclusions
This document specifies general criteria to be applied in the determination of bacterial endotoxins on or
in health care products, components or raw materials using bacterial endotoxins test (BET) methods,
using amebocyte lysate reagents.
1.2 Exclusions
1.2.1 This document is not applicable to the evaluation of pyrogens other than bacterial endotoxins.
Other endotoxin detection methodologies are not included (see B.12).
1.2.2 This document does not address setting specific endotoxin limit specifications.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
bacterial endotoxins test
BET
assay for measuring bacterial endotoxins by combining an aqueous test sample or test sample extract
with Tachypleus amebocyte lysate (TAL) (3.41) or Limulus amebocyte lysate (LAL) (3.28) reagent and
measuring the resulting proportional reaction via visual, turbidimetric or chromogenic techniques (3.3)
3.2
batch
defined quantity of a product intended or purported to be uniform in character and quality produced
during a specified cycle of manufacture
[SOURCE: ISO 11139:2018, 3.21]
1
© ISO 2023 – All rights reserved

---------------------- Page: 7 ----------------------
ISO/FDIS 11737-3:2023(E)
3.3
chromogenic technique
bacterial endotoxins test (BET) (3.1) methodology that quantifies endotoxins on the basis of a measured
colour-producing reaction proportional to the interaction of Limulus amebocyte lysate (LAL) (3.28) and
endotoxin
3.4
control standard endotoxin
CSE
endotoxin standard preparation whose potency has been standardized against the Reference Standard
Endotoxin (RSE) (3.37) for a specific batch of Limulus amebocyte lysate (LAL) (3.28)
3.5
depyrogenation
process used to remove or deactivate pyrogenic substances to a specified level
Note 1 to entry: Pyrogenic substances include bacterial endotoxins.
[SOURCE: ISO 11139:2018, 3.77]
3.6
direct contact
medical device or medical device component that comes into physical contact with body tissue
[SOURCE: ISO 10993-1:2018, 3.6]
3.7
end product
product samples that have completed the entire manufacturing process
Note 1 to entry: For the purposes of this document, end-product testing can be performed prior to sterilization
(pre-sterilization samples) or after sterilization (post-sterilization samples). For limitations see 5.2.6.
3.8
endotoxin
bacterial endotoxin
lipopolysaccharide (LPS)(3.29) component of the cell wall of Gram-negative bacteria that is heat stable
and elicits a variety of inflammatory responses in animals and humans
[SOURCE: ISO 11139:2018, 3.101]
3.9
endotoxin limit
maximum allowable amount of endotoxin present on the product or in a product extraction solution
3.10
endotoxin unit
EU
international unit
IU
standard unit of measure for endotoxin activity initially established relative to the activity contained
in 0,2 ng of the Reference Standard Endotoxin (RSE) (3.37) Lot EC-2 [US Pharmacopeia (USP) standard
reference material]
Note 1 to entry: Currently, the US RSE EC-6, USP Lot G, and the World Health Organization’s primary international
[45]
endotoxin standard (IS) are sub-lots of the same endotoxin preparation, making the EU and IU equal .
2
  © ISO 2023 – All rights reserved

---------------------- Page: 8 ----------------------
ISO/FDIS 11737-3:2023(E)
3.11
end point
most dilute concentration of a test or control solution for which a positive reaction for bacterial
endotoxin is observed
Note 1 to entry: This definition is used for concentration dependent bacterial endotoxin testing, in contrast to
dilution dependent end point methods described in A.6.1.1.
3.12
enhancement
bacterial endotoxins test (BET) (3.1) anomaly in which a non-endotoxin related factor, usually
attributable to a characteristic of the test sample, elicits a test reaction greater than the amount of
endotoxin present
3.13
gel-clot technique
bacterial endotoxins test (BET) (3.1) methodology that quantifies or detects endotoxin on the basis of
a clot-producing reaction proportional to the interaction of Limulus amebocyte lysate (LAL) (3.28) and
endotoxin
3.14
geometric mean end point
antilog of the average of the logarithmic values with respect to the end points (3.11) from replicate
dilution series converted back to a base 10 number used to establish the central tendency or typical
value from a test solution
3.15
health care product
medical device, including in vitro diagnostic medical device, or medicinal product, including
biopharmaceutical
[SOURCE: ISO 11139:2018, 3.132]
3.16
indirect contact
medical device or medical device component through which a fluid or gas passes, prior to the fluid or
gas coming into physical contact with body tissue (in this case the medical device or medical device
component itself does not physically contact body tissue)
[SOURCE: ISO 10993-1:2018, 3.11]
3.17
inhibition
bacterial endotoxins test (BET) (3.1) anomaly in which a non-endotoxin related factor, usually attributable
to a characteristic of the test sample, elicits a test reaction less than the amount of endotoxin present
3.18
method suitability
inhibition/enhancement test
test used to determine whether a particular sample contains interfering factors that diminish its
accuracy by introducing enhancement (3.12) or inhibition (3.17) into the test system
3.19
interference
interfering factor observed in the performance of the test that exceeds the acceptable threshold for
a given bacterial endotoxins test (BET) (3.1) technique (e.g. positive product control that indicates a
detected endotoxin level less than 50 % or greater than 200 % or ±2 lambda)
3.20
intraocular
located or occurring within or administered through the eye
3
© ISO 2023 – All rights reserved

---------------------- Page: 9 ----------------------
ISO/FDIS 11737-3:2023(E)
3.21
interfering factors
non-endotoxin related factor, usually attributable to a characteristic of the test sample, that causes
inhibition (3.17) or enhancement (3.12)
3.22
intravascular
located or occurring within or administered through the heart or blood vessels
3.23
intralymphatic
located or occurring within or administered through a lymph vessel
3.24
intrathecal, adj,
located, or occurring within or administered through the space under the arachnoid membrane of the
brain or spinal cord
3.25
kinetic method
photometric quantitative techniques (turbidimetric or chromogenic) for bacterial endotoxins test (BET)
(3.1)
3.26
LAL reactive material
LAL-RM
Limulus amebocyte lysate reactive material
any non-endotoxin compound that will activate the Limulus amebocyte lysate (LAL) (3.28) clotting
cascade and cause enhancement (3.12)
3.27
lambda
λ
labelled sensitivity of a Limulus amebocyte lysate (LAL) (3.28) gel-clot reagent, expressed in EU/mL or,
for chromogenic or turbidimetric tests, the lowest point (endotoxin concentration) on the referenced
standard curve
3.28
Limulus amebocyte lysate
LAL
reagent extracted from amebocytes taken from hemolymph of the horseshoe crab, Limulus polyphemus,
that reacts with endotoxin, to form a gelatinous clot and is used to estimate endotoxin levels in bacterial
endotoxins test (BET) (3.1) methods
Note 1 to entry: The term LAL is sometimes used to describe Tachypleus amebocyte lysate (TAL) (3.41), as both
are similar lysates that are used in the BET. They also are often generically referred to as “lysate”.
3.29
lipopolysaccharide
LPS
Gram-negative bacterial cell wall component composed of lipid A, a core polysaccharide, and an O-side
chain
3.30
maximum valid dilution
MVD
maximum amount a sample can be diluted, or the total extraction volume used relative to the sensitivity
of a bacterial endotoxins test (BET) (3.1) in which the specified endotoxin limit (3.9) can be detected
4
  © ISO 2023 – All rights reserved

---------------------- Page: 10 ----------------------
ISO/FDIS 11737-3:2023(E)
3.31
medical device
instrument, apparatus, implement, mac
...